There is little doubt that excess glucose flux through the hexosamine biosynthesis pathway (HBP) can cause insulin resistance. Clinical findings support the contention that glucose-induced insulin resistance likely starts years before the onset of type 2 diabetes, even before prediabetes is recognized. Although a mechanism is not known, in vitro data suggest that increased HBP activity increases O-linked N-acetylglucosamine modification of Sp1, leading to transcriptional activation of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. This HBP-induced response increases plasma membrane (PM) cholesterol that impairs insulin-stimulated glucose transporter GLUT4-mediated glucose transport. Inhibition of HBP activity or blockade of O-GlcNAc-modified Sp1 binding to DNA prevents PM cholesterol accumulation and GLUT4/glucose transport dysregulation. These cell culture data support a novel hypothesis that the breakdown of glucose homeostasis in insulin resistance is secondary to increased HBP-mediated cholesterol biosynthesis. The fact excess PM cholesterol is seen in vivo suggests that regulatory mechanisms that protect against cellular cholesterol accumulation/toxicity may be defective in insulin-resistant fat/muscle. In support of this possibility, the HBP-cholesterolgenic response also impairs ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux from insulin-resistant 3T3-L1 adipocytes. Collectively, these data are in accord with recent gene expression studies showing that alterations in a network of cholesterol metabolism genes are associated with T2D risk. Data from cells and tissues suggest PM cholesterol accumulation diminishes cortical filamentous actin (F-actin) important for GLUT4 regulation. Despite this loss of F-actin, preliminary mechanistic studies in insulin-resistant 3T3-L1 adipocytes show GLUT4 storage vesicles (GSVs) are mobilized by insulin to a position just beneath the cholesterol-laden PM but then fail to incorporate and transport glucose. Data suggest that this impairment results from defective phospholipase D1 (PLD1)-mediated production of phosphatidic acid (PA), which is known to promote GSV/PM fusion. This project will determine whether the in vivo increase in PM cholesterol in insulin-resistant fat/muscle is due to HBP-driven Sp1 transcriptional events, and if defective ABCA1 and/or ABCG1-mediated protection against PM cholesterol accumulation occurs exacerbating insulin resistance (Aim 1). With both the regulation of F-actin polymerization and PLD1 activation occurring at cholesterol-enriched caveolae PM microdomains, this project will also determine if excess PM cholesterol-driven defects in these cytoskeletal/membrane GLUT4-regulatory steps are causally linked to insulin resistance (Aim 2). A key postulate of this application is that the development of glucose intolerance in vivo involves a HBP-induced cholesterolgenic response that impairs one or more distal membrane-based mechanisms of GLUT4 regulation. Advancement of this understanding will reshape our understanding of insulin resistance development and identify new therapeutic targets for its prevention and/or treatment.